Miniprep problems

[Paddy]

Hi all,
I did a miniprep today (I was very eager to get some recently cloned vector to take to sequencing).

I'm working with pET21-d in XL1-Blue E.coli. I have done mini-preps in the past without troubles, however I have always disapointed with the yield. So this time I thought that if i make the E.coli really happy in rich media, they should hopefully make a ton of vector for me. I grew them in YPTG.
The ON was exceptional, so much so, that I had to divide the minipreps into 2 just to get a proper resuspension

Following the prep I was specing the water containing what i hoped was my vector and was obtaining 890 ng/uL...1200 ng/uL. Sounds dodgy right? I thought it was an air bubble. But nope nothing, it was all good. I thought optimistically at first, thinking it was only my vector. However, during the prep I had troubles solubilising the DNA in water during the last step (im using promega). It was as if the column was blocked. But I made sure no precipitate from the earlier steps got onto the column.

The better thought I had was to run a gel. So I did and unfortunately I found the lanes were just smears of glowing junk. I originally suspected genomic DNA contamination. Tell me, is this what happened?

Can you grow bacteria for miniprepping in rich media?
My cell lysis solution has sds crashed out, so I heated the bottle in a beaker of warm water. Could the heated solution have wrecked the prep.
Did I overload the prep?

Here's a picture of the gel
flickr.com/photos/37073882@N03/3413551499/

[wangleipurdue]

Hello Paddy,

I doubt that your sample is contaminated by E.Coli genome. The electrophoresis looks similar to me when I got the genome contamination.
I do not think the problem comes from the rich medium but the process when you did the plasmid extraction. I am also new to DNA things, however, to my knowledge, we need to mix the bacteria with P1 buffer (if you use kit from QIAGEN) with lyseblue until you get the blue solution. And after N3 buffer, it should become colorless and you should get some viscous things. I solved my contamination by bacterial genome by paying attention to those two steps. Hope those information can be a little helpful.